Research in the Synaptic Function Unit (SFU) focuses on the elucidation of molecular mechanisms underlying neurotransmitter release and its modulation. We are identifying proteins that regulate SNARE component trafficking, SNARE complex assembly, its structural coupling to calcium sensors, and synaptic vesicle recycling. In past year, we have obtained promising results on the following projects: (1) Synaptic vesicle exo- and endocytotic processes must be effectively coupled to maintain a level of synaptic homeostasis compatible with continuing neurotransmission. One mechanism by which these criteria could be met would require presynaptic proteins that could modulate the dynamics of neurotransmission by directly interacting with the basic machinery of synaptic vesicle exocytosis and endocytosis. Syntaphilin is a neuronal protein that we first characterized as a binding partner of syntaxin-1. Binding of syntaphilin to syntaxin inhibits the binding of syntaxin to SNAP-25 and thus prevents the formation of the SNARE core complex. Functionally, overexpression of syntaphilin in cultured hippocampal neurons inhibits neurotransmitter release; furthermore, injection of the syntaphilin syntaxin-binding peptide into the presynaptic cell body of superior cervical ganglion neurons results in inhibition of neurotransmission. As calcium influx into nerve terminal signals the onset of both synaptic vesicle exocytosis and compensatory endocytosis, we asked whether an analogous inhibitory clamp of the endocytosis protein complex might be present in synapses in which syntaphilin is expressed. We have demonstrated that, in addition to its effects on the synaptic vesicle release machinery, syntaphilin binds to dynamin-1 and inhibits its interaction with amphiphysin, consequently decreases clathrin-dependent, dynamin-mediated transferrin uptake in COS cells and synaptic vesicle recycling in cultured hippocampal neurons. Our findings implicate syntaphilin as an inhibitory modulator of both SNARE-driven exocytosis and dynamin-mediated endocytosis, and suggest a two-fold mechanism by which syntaphilin inhibits neurotransmitter release in neurons. (2) Using the yeast two-hybrid system with syntaxin-1A as bait, we isolated SNAP-29 from a human brain cDNA library. Our studies show that SNAP-29 is present at synapses, interacts directly with syntaxin-1A, competes with a-SNAP for binding to the SNARE complex, and consequently modulates synaptic transmission by inhibiting disassembly of the SNARE complex. It is attractive, therefore, to speculate that SNAP-29 may control the disassembly of the cis-SNARE complex and regulate the availability of individual SNAREs to form new trans-SNARE complexes between opposing membranes, consequently modulating the process of SNARE complex formation and dynamic recycling during membrane fusion events. (3) By using yeast two-hybrid screening with syntaxin-1A as bait, we have also isolated a cDNA encoding the C-terminal domain of DAPK. Expression of DAPK in the adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies demonstrate that DAPK binds to and phosphorylates syntaxin-1A at serine-188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A mutation S188D, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation and is required for synaptic vesicle docking. Our results suggest that syntaxin is a DAPK substrate, and delineate a novel signal transduction pathway by which syntaxin function is regulated in response to intracellular [Ca2+] and synaptic activity. In addition, our finding that DAPK is enriched in synapses opens a new avenue of study to examine the cellular signal mechanisms by which the efficacy of neurotransmission is modulated in response to synaptic activity.